Blocking endothelial apoptosis revascularizes the retina in a model of ischemic retinopathy
Zoe L. Grant, Lachlan Whitehead, Vickie H.Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas
Zoe L. Grant, Lachlan Whitehead, Vickie H.Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas
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Research Article Angiogenesis

Blocking endothelial apoptosis revascularizes the retina in a model of ischemic retinopathy

Abstract

Aberrant, neovascular retinal blood vessel growth is a vision-threatening complication in ischemic retinal diseases. It is driven by retinal hypoxia frequently caused by capillary nonperfusion and endothelial cell (EC) loss. We investigated the role of EC apoptosis in this process using a mouse model of ischemic retinopathy, in which vessel closure and EC apoptosis cause capillary regression and retinal ischemia followed by neovascularization. Protecting ECs from apoptosis in this model did not prevent capillary closure or retinal ischemia. Nonetheless, it prevented the clearance of ECs from closed capillaries, delaying vessel regression and allowing ECs to persist in clusters throughout the ischemic zone. In response to hypoxia, these preserved ECs underwent a vessel sprouting response and rapidly reassembled into a functional vascular network. This alleviated retinal hypoxia, preventing subsequent pathogenic neovascularization. Vessel reassembly was not limited by VEGFA neutralization, suggesting it was not dependent on the excess VEGFA produced by the ischemic retina. Neutralization of ANG2 did not prevent vessel reassembly, but did impair subsequent angiogenic expansion of the reassembled vessels. Blockade of EC apoptosis may promote ischemic tissue revascularization by preserving ECs within ischemic tissue that retain the capacity to reassemble a functional network and rapidly restore blood supply.

Authors

Zoe L. Grant, Lachlan Whitehead, Vickie H.Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas

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Figure 7

ANG2 is not required for vessel reassembly but is required for expansion of reassembled network.

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ANG2 is not required for vessel reassembly but is required for expansion...
(A and B) Representative images of ANG2 (magenta, gray) expression in control and Bak–/– BaxEC/EC retinas exposed to high oxygen for 48 hours followed by return to room air for 12 hours (+ 12 RA) or 24 hours (+ 24 RA). Costained with PECAM1 (cyan). Pink arrows indicate ANG2+ downward sprouts; yellow arrows indicate patches of ANG2+ vessels. Scale bars: 100 μm. (C) Experimental overview of mice analyzed in D–F. (D and E) Representative images and quantification of central retinal vasculature in Bak–/– BaxEC/EC mice subjected to the time course shown in C and treated with isotype control (n = 3) or anti-ANG2 (n = 4). Stained for PECAM1. Scale bars: 500 μm. Student’s 2-tailed t test. (F) Quantification of network fragmentation in the central retina of Bak–/– BaxEC/EC mice subjected to the time course shown in C and treated with isotype control (n = 3) or anti-ANG2 (n = 4). Data for Bak–/– BaxEC/EC mice exposed to 48 hours of high oxygen from Figure 1G are shown for comparison. Student’s 2-tailed t test. (G–I) Representative images and quantification of vascular area in separate layers from the same field of view of the central retinas of control (isotype control, n = 3; anti-ANG2, n = 4) and Bak–/– BaxEC/EC mice (isotype control, n = 4; anti-ANG2, n = 4). Scale bars: 100 μm. Two-way ANOVA with Tukey’s multiple-comparisons test. All data are mean ± SEM. Each circle represents 1 animal.